Herpes simplex virus type 2 mRNA vaccine and preparation method and application thereof

Abstract

The invention relates to a herpes simplex virus type 2 (HSV-2) mRNA (messenger Ribonucleic Acid) vaccine as well as a preparation method and application thereof. The mRNA vaccine encodes the variant of the HSV-2 UL37 protein, the protein structure of the variant is more stable, and a nucleotide sequence corresponding to the variant is optimized by a codon, so that the stability and the protein expression level of mRNA are improved. The mRNA further comprises a 5 '-untranslated region, a 3'-untranslated region and a Poly (A) tail, and is delivered through lipid nanoparticles (LNP). The mRNA vaccine disclosed by the invention can induce stronger specific neutralizing antibody and T cell immune response, and has a good application prospect in prevention or treatment of HSV-2 infection.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to a herpes simplex virus type 2 mRNA vaccine, its preparation method and application. Background Technology

[0002] Herpes simplex virus (HSV) belongs to the herpesvirus family and has two types: HSV-1 and HSV-2, both of which are double-stranded DNA viruses. HSV-2 is mainly transmitted through sexual contact, causing genital herpes. However, this virus can remain latent in the human nervous system for a long time, and can be reactivated under certain triggers, leading to recurrent infections. It is also highly associated with the risk of HIV infection and transmission. Globally, approximately 846 million people aged 15 to 49 have genital herpes infection. Current antiviral drug treatments work by inhibiting viral DNA synthesis, relieving symptoms but with significant side effects and no clear clinical efficacy in suppressing latent infection and recurrence. HSV has evolved multiple mechanisms to evade recognition and clearance by the innate and adaptive immune systems, posing a natural barrier to vaccine development.

[0003] HSV-2 DNA is approximately 152 kb in length and encodes more than 70 proteins. Enveloping glycoproteins gD, gB, gH, and gL all play crucial roles in infecting host cells. However, attenuated vaccines, viral subunit vaccines, and replication-defective virus vaccines targeting these proteins have not yet achieved clinical breakthroughs. Currently, HSV vaccine development faces numerous challenges.

[0004] Traditional vaccines, such as inactivated and subunit vaccines, have weak immunogenicity, inducing only humoral immune responses and poor cellular immune induction, requiring the addition of adjuvants. While live attenuated virus vaccines can induce immune responses, there is a risk of reversal. These vaccines perform poorly in inducing cellular and mucosal immunity, limiting their practical application.

[0005] While mRNA technology platforms have achieved success in COVID-19 vaccines, their translation into HSV-2 vaccines still faces significant challenges. For example, the development of Moderna's mRNA-1608 vaccine against HSV-2 (clinical trial number NCT06033261) has been terminated. This highlights that mRNA vaccines based on existing antigen selection and design strategies may still be unable to overcome the bottleneck of inducing a sufficient protective immune response. This indicates that simply transplanting a technology platform is not a guarantee of success; the selection and optimization of the antigen itself are crucial.

[0006] In summary, the development of vaccines against herpes simplex virus still faces many challenges. Therefore, there is an urgent need to develop mRNA vaccines that target new candidate antigens. By selecting appropriate antigens and optimizing sequences to enhance antigen expression and presentation efficiency, and inducing strong neutralizing antibodies and cellular immune responses, new strategies can be provided for developing safe and effective vaccines. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention provides a herpes simplex virus type 2 mRNA vaccine. By designing a structure-based rational mutation of the HSV-2 immune escape key protein UL37 and combining it with systematic mRNA sequence optimization, the two key issues of antigen expression efficiency and immunogenicity in vivo are synergistically resolved, resulting in significantly enhanced immunogenicity.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect of the invention, a herpes simplex virus type 2 mRNA vaccine is provided, wherein the mRNA of the vaccine encodes a variant of the HSV-2 UL37 protein, the variant comprising one or more mutations selected from: D51A, E52A, D274C, V316E, A442P, V594T, G804Y, and the deletion of amino acids 1-21 at the N-terminus; or the variant is a variant that has at least 90% sequence identity with the sequence containing one or more of the mutations and retains the immunogenicity of the protein.

[0009] Furthermore, the amino acid sequence of the mRNA vaccine is selected from one of SEQ ID NO:4-6.

[0010] Furthermore, the nucleotide sequence of the mRNA vaccine has been codon-optimized and is selected from one of SEQ ID NO:7-9.

[0011] Furthermore, the nucleotides of the mRNA vaccine contain pseudouridine or N1-methylpseudouridine modification.

[0012] Furthermore, the mRNA also includes a 5' untranslated region, a 3' untranslated region, and a Poly(A) tail, and is delivered via lipid nanoparticles.

[0013] Furthermore, the lipid nanoparticles have a particle size of 90-120 nm and a dispersion index (PDI) < 0.20.

[0014] In a second aspect of the invention, a pharmaceutical composition is provided comprising the mRNA vaccine and a pharmaceutically acceptable carrier.

[0015] Furthermore, the pharmaceutically acceptable carrier includes one or more of buffer solutions, adjuvants, osmotic pressure regulators, or stabilizers.

[0016] In a third aspect of the invention, a method for preparing the mRNA vaccine is provided, the method comprising: Using DNA encoding the UL37 protein variant as a template, in vitro transcription was performed to obtain mRNA; The mRNA was purified and then encapsulated with lipid nanoparticles to obtain the mRNA vaccine.

[0017] In a fourth aspect of the invention, the use of the mRNA vaccine or the pharmaceutical composition described herein is provided in the preparation of a medicament for the prevention and / or treatment of herpes simplex virus type 2 infection.

[0018] The present invention has the following advantages and beneficial effects: The herpes simplex virus type 2 mRNA vaccine provided by this invention has achieved significant technical effects at multiple technical levels through innovative antigen design and system optimization, specifically reflected in the following aspects: 1. Antigen expression efficiency is significantly improved. Through rational mutation design based on molecular dynamics simulations, the UL37 protein variant exhibited significantly superior expression levels compared to the wild type in mammalian cell systems. Transfection experiments in Example 2 showed that the mean fluorescence intensity (MFI) of the combinatorial mutant (Mutation-1) in 293T cells reached 68669±3565, significantly higher than that of the sequence-optimized wild type (39214±2574) and the unoptimized wild type (32035±3725).

[0019] 2. Strong and sustained humoral immune response The vaccine induced high levels of specific neutralizing antibodies and total IgG antibodies. Results from Example 4 showed that after booster immunization, all vaccine groups exhibited significant increases in antibody levels, with the combined mutant vaccine (Vaccine-3) inducing the highest antibody levels and an IgG2a / IgG1 ratio close to 1 (range 0.8-1.2), indicating a balanced Th1 / Th2 mixed immune response.

[0020] 3. Full activation of cellular immune response: The vaccine can still effectively induce specific CD4 up to 6 weeks after immunization. + T cells and CD8 + T-cell immune response.

[0021] 4. This invention organically combines antigen mutation design with mRNA sequence optimization and LNP delivery system to produce significant technical synergy: (1) Mutations that enhance antigen stability and codon optimization synergistically improve protein expression levels; (2) The LNP delivery system ensures efficient delivery and intracellular release of mRNA; (3) The overall design achieves balanced activation of humoral immunity and cellular immunity. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 Schematic diagram of HSV-2 UL37 plasmid construction.

[0024] Figure 2 HSV-2 mRNA vaccination experimental plan.

[0025] Figure 3 Humoral immune response to HSV-2 mRNA vaccine; where A is the level of IgG antibody in mouse serum after primary immunization; B is the level of IgG antibody in mouse serum after booster immunization; and C is the level of specific IgG1 and IgG2a antibodies and neutralizing antibodies in mouse serum.

[0026] Figure 4 The Elispot method was used to determine the level of IFN-γ secreted by spleen cells.

[0027] Figure 5 Intracellular factor staining (ICS) was used to detect the percentage of antigen-specific cytokine-positive cells in the total CD4+ T cell population.

[0028] Figure 6 Intracellular factor staining (ICS) was used to detect the percentage of antigen-specific cytokine-positive cells in the total CD8+ T cell population. Detailed Implementation

[0029] The present invention will be described in detail below with reference to specific embodiments and examples, thereby making the advantages and various effects of the present invention more clearly apparent. Those skilled in the art should understand that these specific embodiments and examples are for illustrative purposes only and are not intended to limit the present invention.

[0030] Throughout this specification, unless otherwise specified, the terminology used herein should be understood as having the meaning commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In the event of any conflict, this specification shall prevail.

[0031] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be purchased from the market or prepared by existing methods.

[0032] The overall concept of this invention is as follows: To solve the technical problem of this invention, the overall concept of this invention is as follows: 1. Antigen optimization The HSV-2 genome is approximately 152 kb, encoding over 70 proteins. UL37 is a key viral envelope protein with deamidase activity, capable of directly modifying host innate immune molecules RIG-I and cGAS, blocking the STING pathway, inhibiting type I interferon responses, and aiding viral immune escape. The amino acid sequence of HSV-2 wild-type UL37 (GenBank accession number: LS480640.1, as shown in SEQ. ID No:1) is presented. Our method for screening and constructing HSV-2 UL37 protein stability-enhancing mutants based on molecular dynamics simulation analysis includes the following steps: (1) Molecular dynamics simulation and conformational wave analysis The wild-type HSV-2 UL37 protein was simulated for 100 nanoseconds using GROMACS molecular dynamics software. By analyzing the root mean square fluctuation values, flexible regions with high conformational fluctuations in the protein structure were identified, including residue ranges of 100-150, 400-500, and 900-1000.

[0033] (2) Mutation strategy design and site screening RMSF (Root Mean Square Fluctuation) is a commonly used analytical metric in molecular dynamics simulations to measure the degree of fluctuation of each atom relative to its average position. It reflects the flexibility of different regions of a protein and corresponds to the B factor (temperature factor) in crystallography. Based on RMSF analysis results and combined with protein structural characteristics, one or more of the following strategies can be used for mutagenesis design: A. Introduce proline to enhance rigidity: Select alanine residues in the flexible region and replace them with proline. Use the ring structure of proline to restrict the conformational freedom of the main chain, thereby reducing local flexibility; for example, mutate alanine at position 442 to proline.

[0034] B. Constructing or strengthening disulfide bonds: Introducing new disulfide bonds between cysteine ​​pairs in suitable spatial positions to enhance structural rigidity through covalent cross-linking; for example, mutating aspartic acid at position 274 to cysteine, so that it forms an intramolecular disulfide bridge with the natural cysteine ​​at position 337.

[0035] C. Introducing salt bridges or hydrogen bond networks: In regions with suitable distribution of charged residues, amino acids with opposite charges are introduced through mutations to form new salt bridges or strengthen hydrogen bond interactions; for example, replacing valine at position 316 with glutamic acid to form a stable salt bridge with arginine at position 282.

[0036] D. Enhance hydrophobic interactions: In the hydrophobic core region, replace polar or charged residues with more hydrophobic residues to enhance the internal hydrophobic stacking effect; for example, replace aspartic acid at position 51 and glutamic acid at position 52 with alanine to enhance the stability of the hydrophobic sheet while maintaining the local structure.

[0037] E. Deletion of high-volatility unstructured regions: For N-terminal foldless regions with significantly high RMSF values ​​(such as residues 1-21), delete them entirely without affecting the folding of the core structural domain, in order to improve the overall structural compactness.

[0038] 2. Construction of combined mutants By combining one or more of the above mutation strategies, a UL37 protein variant containing modifications at one or more of the following sites can be constructed: Deletion mutations of D51A / E52A, D274C, V316E, A442P, V594T, G804Y and N-terminal residues 1-21.

[0039] Furthermore, we performed codon optimization on the amino acid sequences of wild-type and variant UL37. By adjusting the GC content and considering both CAI and MFE values, we obtained optimized sequences containing mutations, as shown in SEQ ID NO:7-9, and optimized sequences of UL37 without mutations, as shown in SEQ ID NO:3. The optimized nucleic acid sequences were inserted between the BamHⅠ and SpeI restriction sites of the pVRC vector, which already contained the non-coding region sequence. Specifically, SEQ ID NO:1 is the amino acid sequence of wild-type UL37, and SEQ ID NO:4–6 are the corresponding mutant amino acid sequences; SEQ ID NO:2 is the unoptimized wild-type UL37 nucleic acid sequence, SEQ ID NO:3 is the codon-optimized wild-type nucleic acid sequence, and SEQ ID NO:7–9 are the codon-optimized nucleic acid sequences of the corresponding mutants.

[0040] 3. mRNA vaccine construction Based on the optimized sequence described above, an mRNA vaccine containing the coding sequence of the HSV-2 UL37 variant was constructed. This vaccine also includes essential elements consistent with Pfizer / BioNTech's COVID-19 vaccine BNT162b2, such as a 5' untranslated region (5'UTR), a 3' untranslated region (3'UTR), and a Poly(A) tail, to enhance mRNA stability and translation efficiency.

[0041] To ensure that the mRNA vaccine can be effectively delivered to antigen-presenting cells, lipid nanoparticles (LNPs) are used as the delivery system.

[0042] 4. Verification of humoral and cellular immune responses induced by HSV-2 mRNA vaccine By transfecting 293T cells, we verified that the mutant and sequence-optimized mRNA vaccine significantly increased UL37 protein expression, while the mutation did not significantly affect transfection efficiency. Further animal experiments verified the ability of the mutant and sequence-optimized mRNA vaccine (Vaccine3-5) to induce stronger humoral and cellular immune responses.

[0043] The present application will now be described in detail with reference to embodiments and experimental data.

[0044] Example 1: Design, preparation and analysis of HSV-2 UL37 mRNA 1. HSV-2 mRNA sequence design Based on the sequence of UL37 protein from the HSV-2 isolate (GenBank accession number: LS480640.1) published in GenBank (GenBank: SPT06144.1), codon optimization was performed on wild-type and mutant strains (Table 1, amino acid sequences are shown in SEQ ID NO: 1 and SEQ ID NO: 4-6). This included adjusting the codon bias in mouse and human expression, while considering both expression level (codon adaptation index, CAI) and stability (minimum free-energy change, MFE). The GC content was 50–70%; codon adaptation index (CAI) ≥ 0.85; mRNA secondary structure minimum free energy (MFE) below -1200 kcal / mol. Four optimized nucleotide sequences were obtained, with the mutant sequences shown in SEQ ID NO: 7-9 and the optimized wild-type UL37 sequence shown in SEQ ID NO: 3. The expression vector was pVRC, and the restriction enzyme site was BamH. I and SpeI, the four codon-optimized sequences and the unoptimized wild-type sequence (SEQ ID NO:2, see GenBank) were synthesized and plasmids were constructed, as designed in [reference needed]. Figure 1 .

[0045] 2. Preparation of HSV-2 mRNA stock solution The plasmid was transformed into E. coli and cultured, and positive clones with stable polyA were selected. After expansion culture in shake flasks, the bacterial cells were collected, and after alkaline lysis, neutralization, clarification, concentration and medium replacement, purification, linearization restriction enzyme digestion, and template purification, a high-quality template was obtained for in vitro transcription.

[0046] Using linearized DNA as a template, an in vitro transcription system containing ATP, CTP, GTP, and N1-methyl-pseudouridine-5'-triphosphate with completely substituted UTP was used. The cap analog CleanCap® AG and T7 RNA polymerase were added, and the reaction was carried out at 37°C for 2 to 4 hours to achieve nucleoside modification and co-transcriptional capping. After the reaction, RNase-free DNase I was added to completely degrade the DNA template. The reaction solution was purified by lithium precipitation to obtain the mRNA stock solution. mRNA integrity was determined using a microfluidic parallel capillary electrophoresis system (Fragment Analyzer system 5200, Agilent). mRNA meeting the integrity requirements (>90%) was used in Examples 2 and 3.

[0047] Example 2: In vitro transfection and protein expression analysis of HSV-2 mRNA 1. 293T transfection (1) 293T cells were seeded into 6-well plates at a density of 500,000 cells / well and cultured in 10% DMEM medium to achieve a confluence of 60%-70% at the time of transfection.

[0048] (2) Dilute HSV-2 mRNA and transfection reagent in serum-free medium and incubate at room temperature for 5 minutes. Mix the diluted RNA solution with the diluted transfection reagent and mix gently. Incubate at room temperature for 20 minutes to allow the transfection complex to form.

[0049] (3) Discard the 10% DMEM medium and add the transfection complex dropwise into the cell culture plate, gently shaking the plate to ensure uniform distribution. Incubate the cells in a 37°C, 5% CO2 incubator for 6 hours.

[0050] (4) After the cells adhere, discard the serum-free medium and replace it with 10% DMEM medium, and continue to culture the cells for 24 hours.

[0051] 2. Protein expression detection (1) The cells were fixed with 4% paraformaldehyde for 15 minutes at room temperature. Subsequently, the cells were treated with 0.2% Saponin for 10 minutes to disrupt the cell membrane.

[0052] (2) The cells were incubated with HSV-2 UL37 antibody as primary antibody and FITC-labeled anti-mouse IgG secondary antibody, respectively.

[0053] (3) Collect the treated cells and detect their fluorescence intensity by flow cytometry. The experimental results are shown in Table 1.

[0054] Table 1 - Expression of UL37 protein in 293T cells

[0055] As shown in Table 1, all constructs achieved high-efficiency expression in 293T cells, with a positive cell percentage exceeding 83%, indicating good transfection efficiency and that the mutations did not significantly affect protein synthesis and localization. Regarding mean fluorescence intensity, the Mutation-1 combined mutant exhibited the highest MFI value (68669±3565), significantly higher than the sequence-optimized wild-type WT-2 (39214±2574). The MFI values ​​of Mutation-2 and Mutation-3 were 61566±3988 and 60035±3725, respectively, also significantly better than WT-2, indicating that each mutation strategy contributed to improving protein expression intensity or stability.

[0056] The UL37 protein mutants provided by this invention, particularly Mutation-1 containing multiple combined mutations, exhibit significantly higher expression levels than wild-type UL37 in mammalian cell systems. This result demonstrates that rationally designed structural stability mutations (including disulfide bond introduction, salt bridge construction, proline substitution, and flexible segment deletion) do not affect the correct folding and expression of the protein, and may result in higher overall steady-state expression levels by reducing intracellular degradation, providing a high-performance candidate molecule for subsequent vaccine development.

[0057] Example 3: Preparation of HSV-2 mRNA vaccine 1. To evaluate the formulation characteristics and immunogenicity of the various mRNA vaccine constructs described in this invention, different mRNAs (including wild-type and mutants) were encapsulated in lipid nanoparticles to prepare five groups of vaccine samples, and their key quality parameters were systematically characterized. The vaccine samples included: Vaccine-1: Encapsulates unoptimized wild-type mRNA (WT-1); Vaccine-2: Encapsulated wild-type mRNA with optimized sequence (WT-2); Vaccine-3: Encapsulation of combined mutant mRNA (Mutation-1). Vaccine-4: Encapsulation of combined mutant mRNA (Mutation-2). Vaccine-5: Encapsulates N-terminal deletion mutant mRNA (Mutation-3).

[0058] 2. The specific operating steps are as follows: (1) Lipid nanoparticles were prepared using microfluidic equipment. mRNA was diluted with citric acid solution and lipids were diluted with anhydrous ethanol.

[0059] (2) The microfluidic left pump draws in lipids and the right pump draws in mRNA. The liquid delivery volume and flow rate are kept at left:right = 1:3. The initial liquid is discarded. The crude product is added to 5 times the volume of pure water (that is, the crude product is diluted 6 times) and gently mixed for later use.

[0060] (3) Pour the crude product into an ultrafiltration tube and centrifuge at 2500 rpm, ~755×g, 4℃. The time depends on the centrifugation conditions. Discard the liquid at the bottom until the crude product is completely separated.

[0061] (4) Fill the ultrafiltration tube with purified water, centrifuge and replace it again, and finally gently blow out the concentrated LNP in the ultrafiltration tube and detect the various data of LNP. The results are shown in Table 2.

[0062] Table 2. Key Quality Parameters of mRNA Vaccines

[0063] As shown in Table 2: All vaccine formulations have a pH value within the range of 7.3-7.4, which meets the requirements for physiological compatibility of injectable formulations.

[0064] The nanoparticles have a size distribution between 102 and 114 nm, and the dispersion coefficients are all below 0.20, indicating that the overall particle size distribution of the LNP system is relatively uniform.

[0065] All vaccines showed an encapsulation rate higher than 88%, indicating that the mRNA was efficiently encapsulated within the LNP. mRNA content assays showed consistent loading levels across groups, ranging from 144 to 168 μg / mL.

[0066] The bacterial endotoxin test results were all below 8 EU / mg, meeting the pharmaceutical standards.

[0067] The mRNA-LNP vaccines (Vaccine-3 to Vaccine-5) based on the UL37 mutant developed in this invention are comparable to wild-type mRNA vaccines (Vaccine-1 and Vaccine-2) in key physicochemical and quality control parameters. They all have good formulation characteristics, encapsulation efficiency and endotoxin control level, meeting the quality requirements for preclinical vaccine development.

[0068] Of particular note is that the mRNAs containing the combined stability mutations (Mutation-1, Mutation-2, and -3) did not cause significant changes in particle size, dispersibility, or encapsulation efficiency after being formulated into LNP formulations. This indicates that the sequence changes and codon optimizations did not adversely affect the assembly of nanoparticles and the encapsulation process of mRNA, demonstrating good formulation compatibility and transformation potential.

[0069] Example 4: Evaluation of humoral immune response induced in mice by mRNA vaccine based on UL37 optimized sequence 1. Immunization schedule (see...) Figure 2 ) To comprehensively verify the immunizing effect of the vaccine described in this invention, 6-8 week old female BALB / c mice were randomly divided into 6 groups (n = 5) and immunized via intramuscular injection with the following mRNA-LNP vaccines: Vaccine-1 (encapsulated wild-type UL37 mRNA, WT-1), Vaccine-2 (encapsulated sequence-optimized wild-type UL37 mRNA, WT-2), Vaccine-3 (encapsulated hybrid mutant UL37 mRNA, Mutation-1), Vaccine-4 (encapsulated hybrid mutant UL37 mRNA, Mutation-2), Vaccine-5 (encapsulated N-terminal deletion mutant UL37 mRNA, Mutation-3), and a negative control (empty LNP). All vaccines were administered at equal doses (5 μg mRNA / dose) on day 0 and day 28. Blood was collected from the eyes at specific time points (D14 and D42), and serum was separated to detect antibodies and neutralizing antibodies. The titers of HSV-2-specific total IgG antibodies and their subtypes IgG1 and IgG2a were detected using an ELISA system, and the levels of neutralizing antibodies in serum were also measured. Six weeks after booster immunization, mice in each group were euthanized to obtain spleens for the determination of cellular immunity levels (see Example 5).

[0070] 2. Mouse serum collection (1) Blood was collected from mice using the orbital blood sampling method, with each blood sample containing approximately 50 μL. The collected whole blood was first incubated at 37°C for 1 hour, and then left to stand at 4°C for 1 hour.

[0071] (2) Centrifuge the blood sample at 4000 rpm for 15 minutes, repeating the centrifugation twice to separate the serum. The separated serum samples were aliquoted. One serum sample was placed in a 56°C water bath for 30 minutes to inactivate complement for subsequent neutralizing antibody detection. All serum samples were stored at -80°C.

[0072] 3. Detection of mouse serum antibody IgG (1) Coating: Dilute the purified protein to 5 ug / ml with ELISA coating buffer. Add 100 μL of the diluted protein solution to each well of the ELISA plate. Incubate the plate at 4°C overnight for coating.

[0073] (2) Blocking: Use PBS solution containing 10% goat serum. Add 100 μL of blocking solution to each well of the ELISA plate. Incubate the plate at 37°C for 2 hours.

[0074] (3) Primary antibody incubation: Mouse serum was serially diluted with PBS solution containing 2% goat serum. 100 μL of diluted mouse serum was added to each well of an ELISA plate. The plate was incubated at 37°C for 1 hour.

[0075] (4) Secondary antibody incubation: Dilute HRP-labeled goat anti-mouse IgG (1:10,000) or IgG1 / 2a (1:1,500) with PBS solution containing 2% goat serum. Add 100 μL of the diluted secondary antibody to each well of the ELISA plate. Incubate the plate at 37°C for 1 hour.

[0076] (5) Antibody typing detection: Dilute streptomycin-HRP conjugate antibody (1:1,500) with PBS solution containing 2% goat serum. Add 100 μL of the diluted streptomycin-HRP conjugate antibody to each well of the ELISA plate. Incubate the plate at 37°C for 1 hour.

[0077] (6) Color development: Add 100 μL of single-component TMB color development solution to each well. Develop color in the dark for 2 minutes. Add 100 μL of 2 mol / L H2SO4 solution to each well to terminate the reaction.

[0078] (7) Reading: Use an ELISA reader to read the absorbance value of each well at a wavelength of 450 nm. Use 2.1 times the OD value of the blank well as the cut-off value to determine the positive result.

[0079] 4. Detection of neutralizing antibodies in mouse serum (1) Dilute the serum with DMEM medium at ratios of 1:200, 1:400, 1:800, 1:1600 and 1:3200. Add 100 PFU of HSV-2 MS strain to each well, mix the diluted serum with the virus, and incubate at 37°C for 1 hour.

[0080] (2) Cell infection: The serum-virus complex was added to the prepared Vero cells, and a negative control (DMEM with 2% FBS) and a positive control (HSV-2 MS strain with 100 PFU) were set up. The cells were incubated at 37°C for 2 hours.

[0081] (3) Adding the cover layer: After 2 hours of incubation, add the cover layer, which is a mixture of 2×DMEM and sodium carboxymethyl cellulose in a 1:1 ratio.

[0082] (4) Fixation and staining: Two days later, add 4% cell fixative and fix the cells for 30 minutes. Discard the supernatant and rinse the cells with PBS. Add 0.1% crystal violet staining solution and observe the staining results.

[0083] 4. Results Based on the experimental results Figure 3 show: (1) Two weeks after the initial immunization, the serum of the vaccine group mice was tested and it was found that these mice had produced specific IgG antibodies against the antigen.

[0084] (2) Following the initial immunization, booster immunization significantly increased IgG antibody levels in all vaccine-immunized mouse groups. These booster-immunized mice exhibited significantly higher IgG antibody levels than the control group, indicating that booster immunization has a significant effect on increasing antibody levels. Vaccine-3 antibody levels were the highest, while Vaccine-4 and 5 levels were also significantly higher than in the WT group (related data are shown in [link to relevant data]). Figure 3 middle).

[0085] (3) Two weeks after booster immunization, the levels of specific IgG1 and IgG2a antibodies and neutralizing antibodies in mouse serum were measured. The results showed that the titers of IgG1 antibodies induced in mice in each immunization group were slightly higher than those of IgG2a antibodies. We calculated the IgG2a / IgG1 ratio, which was close to 1 in all vaccine groups (range 0.8-1.2), indicating that the vaccine of the present invention induced a balanced Th1 / Th2 mixed immune response, which is crucial for simultaneously controlling viral replication (Th1-dominant) and providing humoral protection (Th2-dominant).

[0086] Compared with the Vaccine-1 and Vaccine-2 groups, the Vaccine-3 (Mutation-1), Vaccine-4 (Mutation-2), and Vaccine-5 (Mutation-3) groups induced significantly higher titers of HSV-2-specific binding antibodies and neutralizing antibodies after primary and booster immunizations. No specific antibody response was detected in mice immunized with empty LNP in the negative control group.

[0087] These findings indicate that HSV-2 mRNA vaccines based on UL37 and its mutants can induce a relatively balanced Th1 / Th2 immune response. In other words, the vaccine not only effectively stimulates the body to produce neutralizing antibodies, but also achieves a relative balance between inducing Th1 immune responses (mainly associated with IgG2a) and Th2 immune responses (mainly associated with IgG1).

[0088] Example 5: HSV-2 mRNA vaccine induces cellular immune response 1. Preparation of single-cell suspension from mouse spleen (1) Six weeks after booster immunization, mice were euthanized by cervical dislocation. The mice were then disinfected with 75% ethanol.

[0089] (2) Under aseptic conditions, remove the spleen from the mouse. Place a sterile copper mesh into agar plates. Add 4 mL of serum-free 1640 medium to the culture plate. Grind the spleen using a needle until any remaining white connective tissue is completely removed.

[0090] (3) Transfer the ground spleen single-cell suspension to a 15 mL centrifuge tube. Centrifuge at 1000 rpm for 10 minutes at 4°C. Discard the supernatant and resuspend the precipitate in an appropriate amount of serum-free 1640 medium.

[0091] (4) Add 2 mL of erythrocyte lysis buffer and react for 4 minutes. Add 10 mL of 1640 medium containing 10% fetal bovine serum (FBS) to terminate the reaction. Centrifuge at 1000 rpm for 10 minutes at 4°C. Discard the supernatant and resuspend the precipitate in an appropriate amount of 1640 medium containing 10% FBS.

[0092] (5) Add 1 mL of 1640 medium containing 10% FBS and mix the cell suspension thoroughly. Count the cells using a cell counter or hemocytometer.

[0093] 2. IFN-γ ELISpot (1) Dilute the Purified Anti-mouse IFN-γ antibody with 1×PBS at a dilution ratio of 1:200 (50 μL antibody + 10 mL PBS per plate). Add 100 μL of the diluted antibody solution to each well and incubate the plate at 4°C overnight.

[0094] (2) On the second day, discard the coating solution. Add 200 μL of 1640 medium containing 10% serum to each well and incubate at room temperature for 2 hours. Discard the blocking solution after incubation and add another 200 μL of 1640 medium containing 10% serum.

[0095] (3) Add specific antigen-stimulating peptide (400 ng / strip) to each well. Negative control: Add 200 μL of 1640 medium containing 10% serum to each well. Positive control: Add 200 μL of medium containing ionomycin (IONO) and phorbol ester (PMA) to each well.

[0096] (4) Add the diluted mouse single spleen cell suspension to an ELISPOT plate, adding 5 × 10⁻⁶ cells to each well. 5 Cells (50 μL). Incubate the plate at 37°C for 22 hours.

[0097] (5) Discard the cell culture medium. Add 200 μL of pre-cooled sterile water to each well and wash twice, 3-5 minutes each time. Add 200 μL of PBST to each well and wash three times, 2 minutes each time.

[0098] (6) Dilute DetectionAntibody with a 1:250 dilution buffer (40 μL detection antibody + 10 mL dilution buffer / block). Add 100 μL of the diluted detection antibody solution to each well and incubate at room temperature in the dark for 2 hours.

[0099] (7) Add 200 μL PBST to each well and wash 3 times for 2 minutes each time.

[0100] (8) Dilute the streptavidin-HRP solution at a ratio of 1:100 (add 100 μL + 10 mL of diluent to each sample plate). Add 100 μL of the diluted streptavidin-HRP solution to each well and incubate at room temperature for 1 hour.

[0101] (9) Add 200 μL of PBST to each well and wash 4 times for 2 minutes each time.

[0102] (10) Add 200 μL of PBS to each well and wash twice, 2 minutes each time.

[0103] (11) Mix 200 μL of substrate solution with 10 mL of diluent. Add 100 μL of substrate solution to each well and incubate at room temperature in the dark for 20 minutes.

[0104] (12) Stop the colorimetric reaction with running water and dry in the dark. Use an ELISPOT reader to read the number of spots and calculate the amount of IFN-γ secreted per million spleen cells.

[0105] 3. HSV-2 mRNA vaccine induces increased antigen-specific CD4. + / CD8 + T cells (1) Prepare cell suspension, add 2×10 to each tube 6100 cells (200 μL). Add 4 μg of UL37 stimulating peptide pool (200 μL) to each tube. After incubating at 37°C for 30 minutes, add the inhibitor (1 μL inhibitor + 200 μL 1640 medium containing 10% FBS). Continue incubation at 37°C for 12 hours.

[0106] (2) Collect the incubated cells. Centrifuge at 350 g for 5 minutes and discard the supernatant.

[0107] (3) Add 1 mL of DPBS to each flow cytometry tube to resuspend the pellet. Centrifuge at 350 g for 5 minutes, discard the supernatant, and repeat twice. Add 0.6 μL of FVS780 dye to each tube, resuspend the cell pellet with 1 mL of DPBS, and gently vortex to mix. Incubate at room temperature for 15 minutes in the dark. Centrifuge at 350 g for 5 minutes and discard the supernatant.

[0108] (4) Resuspend the cell pellet in 1 mL of PBS solution containing 1% FBS and gently vortex to mix. Centrifuge at 350 g for 5 minutes, discard the supernatant, and repeat twice.

[0109] (5) Add 1 mL of cell staining solution to each tube, centrifuge at 350 g for 5 minutes, and discard the supernatant. Add 250 μL of fixation / permeabilization buffer to each tube to resuspend the cell pellet, and gently vortex to mix. Incubate at 4°C in the dark for 20 minutes. Without discarding the supernatant, add 1 mL of washing buffer directly, gently vortex to mix, centrifuge at 350 g for 5 minutes, discard the supernatant, and repeat twice.

[0110] (6) Add 2 μL of Fc Block antibody and 100 μL of cell buffer to each tube and resuspend the cell pellet. Vortex and incubate at room temperature for 10 minutes.

[0111] Cell surface staining: Add 20 μL of cell staining buffer to each tube, and then add the following antibodies: Table 3. Flow cytometry information related to cell surface markers

[0112] After vortexing, incubate at 4°C for 15 minutes. Washing: Add 1 mL of cell staining buffer to each tube, centrifuge at 350 g for 5 minutes, discard the supernatant, and repeat twice.

[0113] Intracellular antibody staining: Centrifuge at 450 g for 5 minutes at 4°C, and resuspend the cell pellet in 100 μL of washing buffer. Add the fluorophore-conjugated antibody and gently vortex to mix. Table 4. Flow cytometry information related to intracellular cytokines

[0114] Incubate at 4°C for 30 minutes. Without discarding the supernatant, add 1 mL of 1×Perm / Wash buffer, gently vortex to mix, centrifuge at 350 g for 5 minutes, and discard the supernatant. Wash twice with 1 mL of cell staining buffer, centrifuge at 450 g for 5 minutes, and discard the supernatant. Resuspend the cell pellet in 300 μL of cell staining buffer.

[0115] (7) Flow cytometry analysis: Cells were analyzed using flow cytometry to detect the expression of cytokines. Data analysis was performed using FlowJo v10 software.

[0116] 4. Results like Figure 4 As shown, 6 weeks after booster immunization, Vaccine-1-5 significantly increased IFN-γ secretion from mouse spleen cells compared to the control group, indicating that even 6 weeks post-immunization, all five drugs still induced high levels of specific T-cell immune responses in mice. Among them, Vaccine-3 produced the highest level of cellular immunity after immunization.

[0117] Intracellular cytokine staining (ICS) and flow cytometry were further used to evaluate the characteristics of Vaccine-3-5-induced T cell responses. The results showed that after booster immunization, all groups of mice immunized with the vaccine induced the production of CD4+ cytokines, predominantly Th1-type. + T cells and CD8 + T cells secrete cytokines including IFN-γ, IL-2, and TNF-α, while the secretion of the Th2-type cytokine IL-4 is relatively low (see relevant data). Figure 5 , Figure 6 Among them, Vaccine-3 showed the best induction effect, inducing high levels of IFN-γ and IL-2. These results indicate that the HSV-2 mRNA vaccine based on the UL37 mutant can effectively induce specific CD4 production in mice. + T cells and CD8 + T-cell immune response. In mice immunized with Vaccine-3, the spleens of these mice simultaneously secreted double-positive CD8+ cells containing both IFN-γ and TNF-α. + The proportion of T cells was significantly higher in the HSV-2 infection model than in other groups. These pluripotent T cells are typically associated with stronger antiviral activity and immune memory. In the HSV-2 infection model, IFN-γ and CD8+... +T cell-mediated cytotoxicity is widely recognized as the most critical mechanism for controlling viral replication and clearing infected cells. The high-quality T cell response elicited by the vaccine of this invention is highly consistent with known protective immune response profiles, strongly suggesting its potential to clear viruses and control disease progression.

[0118] In this invention, the in vitro protein expression level (MFI value in Table 1) and the in vivo immune response intensity ( Figure 3-6 Antibody titer, number of spleen cells secreting IFN-γ, and CD4+ positive for specific cytokines in the sample. + or CD8 + There was a clear positive correlation between the percentage of T cells and the expression level: Mutation-1 (Vaccine-3), with the highest expression level, induced the strongest response across all immune markers, and maintained a high level of cellular immunity for 6 weeks post-immunization, significantly higher than Vaccine-4 and Vaccine-5; while wild-type UL37 sequences (Vaccine-1, 2) with lower expression levels than mutants also exhibited weaker immune responses. This correlation confirms that our technical approach of improving antigen protein stability to obtain higher expression levels can be directly and effectively translated into superior immunoprotective efficacy.

[0119] Finally, it should be noted that the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make further changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention. Clearly, those skilled in the art can make various alterations and modifications to the invention without departing from its spirit and scope. Thus, if these modifications and modifications of the invention fall within the scope of the claims and their equivalents, the invention also intends to include these modifications and modifications.

Claims

1. A herpes simplex virus type 2 mRNA vaccine, characterized in that, The vaccine's mRNA encodes a variant of the HSV-2 UL37 protein, the variant comprising one or more mutations selected from: D51A, E52A, D274C, V316E, A442P, V594T, G804Y, and the deletion of amino acids 1-21 at the N-terminus; or the variant is a variant that has at least 90% sequence identity with the sequence containing one or more of the mutations and retains the immunogenicity of the protein.

2. The mRNA vaccine according to claim 1, characterized in that, The amino acid sequence of the mRNA vaccine is selected from one of SEQ ID NO:4-6.

3. The mRNA vaccine according to claim 1, characterized in that, The nucleotide sequence of the mRNA vaccine has been codon-optimized and is selected from one of SEQ ID NO:7-9.

4. The mRNA vaccine according to claim 1, characterized in that, The nucleotides of the mRNA vaccine contain pseudouridine or N1-methylpseudouridine modification.

5. The mRNA vaccine according to any one of claims 1-4, characterized in that, The mRNA also includes a 5' untranslated region, a 3' untranslated region, and a Poly(A) tail, and is delivered via lipid nanoparticles.

6. The mRNA vaccine according to claim 5, characterized in that, The lipid nanoparticles have a particle size of 90-120 nm and a dispersion index (PDI) of <0.

20.

7. A pharmaceutical composition, characterized in that, The mRNA vaccine comprising any one of claims 1-6 and a pharmaceutically acceptable vector.

8. The pharmaceutical composition according to claim 7, characterized in that, The pharmaceutically acceptable carriers include one or more of the following: buffer solutions, adjuvants, osmotic regulators, or stabilizers.

9. A method for preparing the mRNA vaccine according to any one of claims 1-8, characterized in that, The method includes: Using DNA encoding the UL37 protein variant as a template, in vitro transcription was performed to obtain mRNA; The mRNA was purified and then encapsulated with lipid nanoparticles to obtain the mRNA vaccine.

10. The use of the mRNA vaccine of any one of claims 1-6 or the pharmaceutical composition of any one of claims 7-8 in the preparation of a medicament for the prevention and / or treatment of herpes simplex virus type 2 infection.

Images